Production process of recombinant placental growth factor

ABSTRACT

Process for extracting and purifying the recombinant Placental Growth Factor (PLGF) expressed in inducible prokaryotic expression systems comprising the following steps: I) fermentation of the bacterial cells, II) extraction and purification of the inclusion bodies, III) renaturation of the expressed protein, IV) ion-exchange chromatography, V) reverse-phase chromatography.

FIELD OF THE INVENTION

The present invention relates to a process for extracting and purifyingthe recombinant Placental Growth Factor from genetically modified cells.

STATE OF ART

The Placental Growth Factor (PLGF) is a homodimeric glycoprotein with astructure similar to the Vascular Endothelial Growth Factor (VEGF). Thecomplete polynucleotide sequence codifying the PLGF protein wasdescribed by Maglione and Persico in patent EP-B-550 519 (WO-A-92/06194)claiming Italian priority of 27.09.1990. Alternative processes ofsplicing the ARN of PLGF generate three homologue forms of the PlacentalGrowth Factor, precisely PLGF-1, PLGF-2 and PLGF-3, having differentpolypeptide sequences, and all described in literature.

The above-mentioned patent also describes a method for producing thePLGF factor comprising the use of an inducible prokaryotic expressionsystem characterised by host cells modified with an expression vector,in which the human PLGF gene is integrated under the control of aninducible promoter (directly or indirectly). After inducing the PLGFexpression with appropriate activator, the cells are incubated, isolatedand submitted to lysis.

The so-obtained raw lysate is a complex mixture of proteins containinglow quantities of the expressed PLGF protein and having low specificactivity. In fact, in the known process, the protein expression isinduced in cultures containing low cellular density, as it is evidentfrom the low optical density at time of induction, that is between 0.2and 0.6 OD at 600 nm. Furthermore, the process described in thepreceding document does not comprise additional purification stages ofthe expressed protein. For this reason, the lysates obtained accordingto the application WO-A-92/06194 are inappropriate as such to be useddirectly in the preparation of medicaments.

A more complex method for purifying the same placental factor isenvisaged by Maglione et al. in “Il Farmaco” 55 (2000), pages 165 to167. Nevertheless, the method disclosure merely gives a simple listingof known applicable techniques, without describing conditions andexperimental details thereof, which are essential for obtaining the PLGFprotein in the purity and quantity necessary for a pharmaceutical use.

The scope of the present invention is to provide a new method forextracting and purifying the recombinant PLGF expressed in bacterialcells, allowing to obtain PLGF at high level of purity and with yieldssuitable to be industrially used in the preparation of medicaments.

A further scope of the invention is to obtain the PLGF protein inessentially active form (greater than 98.5%), that is mainly composed bydimeric (not less than 70%) and multimeric forms and containing residuesof the monomeric form (little or not active) not greater than 1.5%.

SUMMARY OF THE INVENTION

The invention is based upon the identification of a sequence ofpurification techniques particularly appropriate for the extraction andpurification of human PLGF expressed in bacterial cells. The inventionis further based upon the determination of the optimum operativeconditions with respect to the single techniques and to thechemical-physical features of the substance to be purified.

Then, object of the present invention is a process for extracting andpurifying the recombinant Placental Growth Factor (PLGF) expressed bymeans of an inducible prokaryotic expression system comprising the stepsof: I) fermentation of the bacterial cells, II) extraction andpurification of the inclusion bodies, III) renaturation of the expressedprotein, IV) ion-exchange chromatography, V) reverse-phasechromatography, and optionally VI) final stages of ultrafiltration,formulation and lyophilization. According to such method, thefermentation (step I) is performed until obtaining a high bacterialdensity in the medium, as it is shown by the high optical density in themedium, before proceeding with the induction step. The step (II)comprises bacterial lysis, rupture of DNA and isolation of the inclusionbodies. The renaturation of the expressed protein (step III) is obtainedby solubilizing the inclusion bodies in denaturant buffer, and bytransforming, at least partially, the expressed protein in the dimericform. At last, in the step (IV) and (V) the dimeric and multimeric formsof the expressed protein are separated from the monomeric form andisolated in pure form, to be subsequently ultrafiltrated and lyophilisedin presence of usual lyophilisation and formulation additives.

A specific object of the invention is the above-mentioned process forextracting and purifying the PLGF-1 protein of human origin, butsubstantially valid also for PLGF-1 of animal origin.

The claimed process advantageously allows the obtaining of productionyields of expressed protein from 30 to 50 times higher than the yieldsobtained according to the method described in the preceding state ofart. The claimed process furthermore allows the obtaining of the highlypure protein, with high specific activity and in substantially dimericform.

Further object of the invention is the active Placental Growth Factorobtainable by means of the process of the invention, free from anyresidual protein or other bacterial contaminant and containing residuesof monomeric form not higher than 1.5%.

DESCRIPTION OF THE FIGURES

FIG. 1: The figure shows the results obtained by electrophoresisSDS-PAGE under reducing conditions for monitoring step I. Pre1 and Pre2represent the pre-induction checks, prepared as follows. Soon before theinduction, about 0,064 units of optical absorption measured at 600 nm(OD600) are taken and diluted 5 times with water. Of this dilution 20 μlare taken which are added to 20 μl of reducing buffer and submitted toboiling. 20 μl of this last solution are loaded onto the SDS PAGEmatrix. Post1 and Post2 represent the post-induction checks prepared asabove. M designates the mixture of markers of molecular weight. In thepost-induction columns (Post1 and Post2) a band is noted just above theindicator of molecular weight 14.3 substantially absent in thepre-induction columns (Pre1 and Pre2) corresponding to the PLGF-1protein expressed and segregated within the inclusion bodies.

FIG. 2: The figure shows the monitoring chromatogram of theanionic-exchange chromatography on Q-Sepharose Fast Flow resin. X-axisshows the elution volume (ML), y-axis shows the optical absorption units(OD). The first elution peak obtained by eluting by 20% with buffer B(NaCl 200 mM) corresponds to the PLGF-1 protein in substantially dimericform, but it comprises impurities and the monomeric form. The followingpeak, eluted by 100% with buffer B (NaCl 1M) contains impurities whichare eliminated.

FIG. 3: The figure shows the monitoring chromatogram of the firstelution stage in reverse-phase chromatography on RP Source 30 resin.X-axis shows the elution volume (ml), y-axis shows the opticalabsorption units (OD). The first abundant elution peak corresponds tothe various impurities, which do not bond to the resin. The secondelution peak corresponds to the PLGF protein in monomeric form elutedunder isochratic conditions (experimentally found at about 10%-15% ofbuffer B).

FIG. 4: The figure shows the monitoring chromatogram of the secondelution stage in reverse-phase chromatography on RP Source 30 resin.X-axis shows the elution volume (ml), y-axis Shows the opticalabsorption units (OD). The elution peak corresponds to the PLGF proteinin dimeric-multimeric form.

FIG. 5: The figure shows the results obtained in electrophoresis onSDS-PAGE for the final monitoring of the whole process. Prerefol andPostrefol represent the checks preceding and following the renaturationof the expressed protein (step III). QFF represents the peak eluted fromthe Q Sepharose fast flow resin, containing the protein mainly indimeric form. Mon represents the peak containing the monomeric form. Dimrepresents the peak eluted in the second substep of the reverse-phasechromatography on RP Source 30 resin. It is noted that before therenaturation, the expressed protein is mainly in monomeric form. Afterthe renaturation, part of the protein is in dimeric form. The followingpurification on QFF and RF 30 chromatography allow the obtaining ofPLGF-1 protein with high purity degree.

DETAILED DESCRIPTION OF THE INVENTION

The genetic modification of the bacterial host cells is described byMaglione et al. in the preceding patent EP-B-0550519 (WO-A-92/06194).For this purpose, bacterial cells are transformed introducing of anexpression vector comprising an insert corresponding to the human genecoding for PLGF-1 factor. The complete gene sequence is known inliterature and it is freely accessible. A plasmid containing suchsequence was deposited with the ATCC under accession number ATCC No40892. The expression is performed under the control of the system ofRNA polymerase of T7 phage and it is induced with IPTG(isopropyl-β-D-tiogalactopyranoside).

Nevertheless, other inducible prokaryotic expression systems may beutilised. Examples of such systems, obtainable on the market, arerepresented by:

1) pBAD expression system (In vitrogen BV) wherein the synthesis of aprotein is placed under the control of the araBAD promoter and it may beinduced in different strains of E. Coli by means of arabinose.

2) T7 Expression System (In vitrogen BV or Promega) wherein thesynthesis of a protein is controlled by the promoter of RNA polymeraseof T7 phage and it may be induced by means of lactose or the analoguesthereof (IPTG). In this case it is required the use of E. Coliderivatives of DE3 (Bl21(DE3) or JM109(DE3)) type containing, namely, acopy of the gene of Rna polymerase of T7 phage placed under the controlof a lactose-inducible promoter.

3) Trc expression system (In vitrogen BV) wherein the synthesis of aprotein is placed under the control of the trc hybrid promoter. Suchpromoter has been obtained by melting the trp promoter and the lacpromoters and it may be induced in different strains of E. Coli by meansof lactose or the analogues thereof (IPTG).

4) Tac expression system (Amerham biosciences) wherein the synthesis ofa protein is placed under the control of the tac promoter. In thissystem the protein synthesis is induced in strains of E. Coli lacIq(type JM105) by means of lactose or the analogues thereof (IPTG).

5) P_(L) expression system wherein the synthesis of a protein is placedunder the control of the PL promoter and it may be induced by addingtryptophan. In this case it is required the use of E. Coli derivatives(GI724) containing a copy of the codifying gene for the cI repressor ofthe Lambda phage placed under the control of a tryptophan-induciblepromoter.

Step I: Fermentation and Induction

The first stage of the claimed process consists in the fermentation of afunctionally-modified bacterial strain equivalent to the straindescribed in the preceding European patent (above). In a preferredembodiment the micro-organism is a derivative of Escherichia Colimodified with an expression plasmid comprising the human gene of PLGF. Apreferred micro-organism is the one called [B12(DE3)pLysS PLGF-1]obtained by integrating in the commercially available strain[B12(DE3)pLysS] (Promega Corporation USA) the gene of the human PLGF-1.The present invention, nevertheless, is not limited to the human PLGF-1factor, but it also relates to the one of animal origin (monkey, mouse,rabbit etc.). The present invention is not limited so much the less tothe use of a E. Coli derivative, but it includes the use of anyprokaryotic micro-organism susceptible to be genetically modified andable to express heterologous proteins under the form of inclusionbodies.

The strains utilised as inoculum in the process of the invention arekept before using them in the lyophilised form to preserve theexpression capacity thereof. Upon use, the lyophilised material isbrought again in solution by utilising an appropriate buffer.

Although there is a wide range of known culture media available on themarket and which may be effectively used, the fermentation stepaccording to the invention is preferably performed in a medium free fromany material of animal or human origin in order to avoid any infectionrisk. Yeast's extracts (Difco) added with one or more suitableantibiotics represent the most suitable means for the process. In thepreferred embodiment a medium is used which has been obtained by mixingunder sterility conditions a first solution (A) containing yeast'sextracts, glycerol and ammonium sulphate with a second solution (B)containing a phosphate buffer. The mixture is then integrated withampicillin and chloramphenicol or equivalent antibiotics. Appropriateantibiotic concentrations are from 50 to 300 μg/ml of ampicillin,preferably from 100 to 200 μg/ml and from 10 to 100 μg/ml ofchloramphenicol, preferably from 30 to 40 μg/ml.

The fermentation step may be preceded by a preinoculum step wherein thelyophilised micro-organism is suspended in the medium and submitted toconsecutive incubation and dilution steps aimed at having in culture theoptimum quantity of micro-organism cells. Preferably, the micro-organismis incubated for one night at 37° C., then diluted and incubated againfor some hours. The chosen pre-inoculum volume is subsequentlycentrifuged, suspended again in the culture solution enriched withampicillin and inoculated in the fermentation vessel for thefermentation step.

The fermentation is performed in the above-mentioned medium added withampicillin and chloramphenicol at the temperature suitable for themicro-organism, usually at about 37° C., in presence of a percentage ofdissolved O2, with respect to the saturation with air, from 20% to 40%,preferably 30%. The pH during fermentation is kept at neutral or weaklyacid values (6.4 to 7.4). Furthermore, since the fermentation processtakes place under stirring, antifoam agents are preferably to be used.

The fermentation progress is accompanied by the increase in the opticaldensity of the medium. For this reason, the optical density is theparameter utilised according to the invention to monitor the progressdegree of the fermentation. Readings at 600 nm are particularlyappropriate.

Essential feature of the invention is the high cellular density achievedin the culture at time of the expression induction. Optical densities at600 nm (OD600) from 1 to 50 may be achieved thanks to culture media ofthe invention. Densities higher than 18, nevertheless, are preferred toobtain the high production levels typical of the claimed method.Densities between 16 and 20 are particularly preferred to induce theproducing bacterial strain and gave optimum results. The fermentation,then, is kept at the above-mentioned conditions until achieving suchvalues of optical density, then one proceeds to induce the proteinexpression.

Any agent or chemical-physical condition able to induce in the cells ofthe used micro-organism the machinery of expression of the heterologousprotein may be advantageously utilised. In the specific case wherein thebacterial strain BL21(DE3)pLysS modified with an expression plasmidcontaining the promoter of T7 phage is used, the expression is inducedwith lactose or the derivatives thereof, such asisopropyl-β-tiogalactopyranoside (IPTG) with a proper concentration,namely about 1 mM. The induction duration may vary according to need.Good results are obtained for periods of some hours, preferably from 3to 4 hours; in the optimum process the induction is kept for 3 hours and20 minutes by using a percentage of dissolved O2 equal to about 10%.

Cell samples are taken before and after induction and submitted toanalytical techniques of control such as electrophoresis on SDS-PAGE, todetermine the induction outcome.

When the protein expression reaches the desired levels, the culture iscentrifuged and the cells are moved to the following step.

Step II: Extraction and Purification.

The expressed heterologous protein in bacterial strains is segregatedinside the cell itself in the form of inclusion bodies. Therefore, theprocess of the invention provides passages of lysis of the cells,rupture of the extracted nucleic material (DNA) and recovery and washingof the inclusion bodies.

The cells are washed, although not necessarily, and suspended insolutions containing emulsifier agents in appropriate concentration,preferably Triton X100 in concentrations from 0.5% to 1%, then they aresubmitted to lysis of the cellular membrane. The lysis process may beperformed by means of freezing/thawing, French Press, sonication orother similar known techniques. Nevertheless, the preferred method forthe bacterial strain BL21(DE3)pLysS is the freezing/thawing method,which in the most preferred embodiment is repeated at least for twoconsecutive cycles. After the mechanical lysis, the lysis stage iscontinued for a few minutes in the lysis solution at room temperatureunder stirring.

The release in the lysis medium of the inclusion bodies is accompaniedby the release of micro-organism different components and cellularsubstances, above all the nucleic materials. These substances couldinterfere with and jeopardise the following protein purificationprocess. Therefore, the suspension/solution obtained by lysis issubmitted to rupture of such nucleic material, specifically DNA, bymeans of enzymatic agents, such as DNAse (natural or recombinant such asthe Benzonase), chemical agents, such as deoxycholic acid, orphysical-mechanical agents, such as sonication or high energy stirringby means of blades, for example, in a mixer. The rupture of DNA, carriedout for example in a mixer, is performed on lysed cells re-suspended inappropriate volumes of washing solutions containing chelating anddeterging agents, for example EDTA and Triton X100. It is preferablyrepeated for more cycles, preferably 2, alternated with stages ofdilution, in washing solution, centrifugation and elimination of thesupernatant in order to remove components and cellular substances fromthe fraction containing the inclusion bodies.

Step III Renaturation (Refolding) of the Protein

The fraction containing the purified inclusion bodies of PLGF-1 is thensolubilised in denaturing buffer containing known denaturant agents suchas urea, guanidine isothiocyanate, guanidine-hydrochloride. Preferably,the denaturant solution is a urea solution in denaturant concentration,for example 8M. In order to accelerate the solubilisation process, thefraction may advantageous be submitted to homogenisation or sonication.After solubilizing the inclusion bodies, the solution is diluted withthe same denaturant buffer until obtaining an optical density measuredat 280 nm of about 0.8 (OD280 0.8). Subsequently, the solution isfurther diluted with a dilution buffer until 0.5 OD280. Suitabledilution solutions contain salts and polyethylene glycol (PEG) and havebasic pH (about 8). The renaturation of the PLGF-1 protein in dilutedsolution is obtained by adding to the solution appropriateconcentrations of oxidising/reducing pairs, followed by an incubation of10 to 30 hours, preferably 18 to 20 at a temperature of 10° C. to 30°C., preferably 20° C., under stirring. Examples of such pairs are:Cystine/Cysteine, Cystamine/Cysteamine,2-hydroxyethyldisulphide/2-mercapto-ethanol. Preferred example ofoxidising/reducing pair is the glutathione in its oxidised and reducedforms, respectively at concentrations between 0.1 mM and 2.5 mM(preferably 0.5 mM) and between 0.25 mM and 6.25 mM (preferably 1.25mM). By means of renaturation, the PLGF-1 protein expressed essentiallyin monomeric form is partially brought back to the dimeric form (FIG.5).

Step IV: Anionic-exchange Chromatography

The solution coming from the preceding step, preferably throughcentrifugation and/or filtration and containing the protein in mainlymonomeric and partially dimeric form, is loaded onto anion-exchangeresin in order to enrich the mixture with the dimeric form and to purifyit from bacterial contaminants. Any commercially available matrixsuitable for anion-exchange chromatography may be likewise used to theextent that its features of capacity, loading and flow speed be similarto those of the Q Sepharose Fast Flow resin (Amersham biosciences),apart from being suitable for an industrial process. In a preferredembodiment a high-flow resin is used, for example Q-sepharose Fast Flow(Amersham biosciences) or equivalent. The resin is washed andequilibrated with solutions having low ionic strength. An example ofsuch solution comprises ethanolamine-HCl pH 8.5 with low or absent saltcontent. The same solution may be utilised for loading, absorbing andwashing the protein mixture to be purified. The used resins allowloading of large volumes of protein solution with ratios Volumeloaded/Volume column varying from 1:1 to 10:1. Ratios Vol./Vol. next to10:1 are preferred since they allow optimising the use of the column.However, ratios higher than 10:1 are to be avoided since, due to thesaturation of the adsorbing capacity of the matrix, they lead to highloss in the dimeric form of the protein.

Whereas the PLGF-1 protein in monomeric form already percolates in thestages of washing with low ionic strength, the elution of the dimericand multimeric forms is obtained by increasing the ionic strength of thestarting solution. Such increase is obtained by mixing the equilibrationsolution with increasing and pre-established percentages of a secondsolution containing NaCl 1M. In a preferred embodiment, the protein indimeric form is eluted with solutions containing from 15% to 25% of NaCl1 M solution, which corresponds to a NaCl concentration from 150 to 250mM. In the best embodiment, the protein is eluted in isochraticconditions at NaCl concentration of 200 mM. The elution of the variousspecies is automatically monitored by measuring the optical absorptionat 280 nm (FIG. 2). The collected fractions containing the PLGF-1protein in dimeric form are subsequently controlled by electrophoresisSDS-PAGE (FIG. 5). Advantageously, the whole chromatography process isautomatically performed by a computerised system operating under thecontrol of a suitable programme, for example the Software FPCL Directorsystem (Amersham biosciences).

Step V: Reverse-phase Chromatography

The fractions coming from the preceding step containing the PLGF-1protein enriched with the active forms are collected, diluted withappropriate buffer and loaded onto an reverse-phase chromatographycolumn in order to further purify the protein in active form. Thequantity of loaded solution corresponds to OD280 between 4.5 and 5.5 permillilitre of chromatographic matrix. Such quantities are to beconsidered maximum quantities.

Any commercially available chromatographic matrix suitable for theintended use may be utilised to the extent that its features of loadingcapacity and flow speed are compatible with the process requirements. Ina preferred embodiment, a resin is used having such bead-sizes so as toguarantee the best exploitation of the absorbing capability togetherwith the easiness in packing the column itself. Examples of suchmatrixes are the RP Source 15 or RP Source 30 (Amersham biosciences)resins. All the solutions for equilibration, loading, resin washing andelution are hydro-organic solutions comprising different percentages oforganic solvent. Examples of such solutions are solutions comprisingethanol, methanol or acetonitrile. Preferably, hydro-alcoholic solutionscomprising increasing percentages of ethanol are utilised. In anembodiment of the invention appropriate quantities of two buffersolutions are mixed, the former comprising buffer A, i.e. ethanol 40%and TFA (trifluoroacetic acid) 0.1%, the latter comprising buffer B,i.e. ethanol 70% and TFA 0.1%.

The protein material loaded onto the resin and properly washed is theneluted through an elution process comprising two subsequent stageswherein elution solutions containing an increasing gradient of organicsolvent are utilised. The first stage is performed under conditions ofrising gradient of organic solvent until obtaining the elution peak ofthe monomeric form. Such gradient is obtained by adding the buffer B tothe buffer A in percentages from 4% to 40%, with an increasing rate ofbuffer B of 3% for each eluted column volume. As soon as the elutionpeak corresponding to the monomeric form of the protein appears, theelution is continued under isochratic conditions until exhaustion of theelution peak of the monomeric form. The so-set isochratic conditionscause the largest possible separation of the chromatographic peakscorresponding to the two monomeric and dimeric forms and, then, the bestobtainable resolution for a process of industrial, and not analyticaltype. The second stage is performed again under condition of increasinggradient of organic solvent until whole elution of the protein mainly indimeric form is achieved. In this second stage, the gradient is obtainedby adding the buffer B to the buffer A in percentages from 10% to 100%,with an increasing rate of buffer B of 40.9% for each eluted columnvolume. The elution of the various forms of PLGF-1 protein isautomatically monitored by measuring the optical absorption at 280 nm(FIG. 3 and FIG. 4). The collected fractions containing the PLGF-1protein essentially in dimeric form are subsequently controlled byelectrophoresis SDS-PAGE (FIG. 5). Advantageously, the wholereverse-phase chromatography process is automatically performed by acomputerised system operating under the control of a suitable programme,for example the Software FPCL Director system (Amersham biosciences).

The results of the electrophoresis show that PLGF-1 protein obtainedfrom the second stage of the reverse-phase chromatography is in highlypure active form, namely it comprises the protein in dimeric andpartially multimeric form, but it is essentially free from anycontamination of the monomeric form. The so-obtained product comprisesnot less than 98.5% of active form, preferably not less than 99.5%,wherein not less than 70% is in dimeric form. The residual of monomericform is not higher than 1.5%. The protein in active form is obtained inaverage amounts of 160 mg per litre of bacterial culture. The pureprotein obtained according to the above-described method may besubmitted to additional working stages such as ultrafiltration onmembrane. In this case the product is filtered on membrane havingcut-off limit lower than, or equal to 30 kD and it is submitted todiafiltration against TFA acidulated water until a dilution factor of1:106. The so-obtained final product may be properly formulated withlyophilisation additives and lyophilised to keep its best biologicalactivity.

The invention is here below described by means of examples having,however, only illustrating and not limiting purposes.

EXAMPLE 1 Fermentation

The following procedure relates to the method of fermentation andinduction of the genetically modified micro-organism (MOGM)[Bl21(DE3)pLysS PlGF-1] in a fermentation vessel using 1 mM IPTG.

Materials: Solution SBM constituted by: Solution A (per 1 liter) Bactoyeast extract (Difco) 34 g Ammonium sulphate 2.5 g Glycerol 100 ml H2Oq.s. at: 900 ml Solution B (10 X) (per 100 ml) KH2PO4 1.7 g K2HPO4 −3H2O20 g, or K2HPO4 15.26 g H2O q.s. at 100 ml

The solutions A and B are separately autoclaved and mixed upon use understerile conditions. Alternatively, the solutions A and B are mixed andfiltered under sterile conditions.

IPTG 200 mM (200X) is produced by dissolving 5 g of pure substance in100 ml of distilled water. The solution is filtered by means of 0.22-μmfilters, subdivided into aliquots and frozen at −20° C.

The utilised antifoam agent is Antifoam O-10 (not siliconic) Sigma CatA-8207.

The used bacterial strain is [BL21pLysS PlGF-1 WCB] (working cell bank).

Preinoculum: A tube of lyophilised genetically modified micro-organism(MOGM) WCB is taken and it is suspended in 1 ml of SBM+100 μg/mlAmpicillin+34 μg/ml of chloramphenicol.

The suspension is diluted in 30 ml of SBM+100 μg/ml Ampicillin+34 pg/mlof chloramphenicol.

The suspension is incubated at 37° C. for one night (O/N). The day afterthe 30 ml of the O/N culture are diluted in 800 ml of SBM+100 pg/mlAmpicillin+34 μg/ml of chloramphenicol and they are subdivided into 41-liter Erlenmeyer flasks, each containing 200 ml.

The content of each flask is incubated at 37° C. for 24 hours. Thecontent of the 4 flasks is mixed and the OD600 are read by diluting 1/20in water (50 μl+950 μl of water).

An established volume of preinoculum is then centrifuged for 10 min. at7.500×g at 4° C. in sterile tubes.

The bacteria are then re-suspended in 20 ml of SBM+200 μg/ml ofAmpicillin+10 μg/ml chloramphenicol per each litre of fermentation bystirring at 420 rpm at R.T. for 20 minutes. At the same time thefermentation vessel is prepared and the oxygen probes are calibrated.

The oxygen probes are calibrated at 37° C. temperature at 0% withnitrogen, then at 100% with air without antifoam under stirring at 600RPM.

The fermentation is carried out under the following experimentalconditions:

Medium: SBM+200 ug/ml of ampicillin and 10 μg/ml of chloramphenicol

Temperature: 37° C. % dissolved O2: 30% (with respect to saturation withair) pH: from 6.4 to 7.4.

Antifoam: 1:10 is diluted in water; strongly stirring before adding itin quantities of 140 μl per 750 ml of medium.

Induction:

The induction is carried out under the following experimentalconditions:

OD600 of induction: 16–20. Inducing agent: IPTG 1 mM final. % dissolvedO2: 10% (with respect to saturation with air). Induction length: 3 hoursand 20 minutes.

Just before the induction 20 μl of bacteria are taken, added to 80 μl ofwater and kept for the pre-induction check.

At time of induction, IPTG is added to the final concentration of 1 mM.

The percentage of dissolved O2 is brought to 10%.

At the end of the induction the final OD600 are read and the overallvolume is measured.

Then, 10 μl of bacteria are taken, added to 90 μl of water and kept forthe post-induction check.

The induction is controlled by way of a SDS-PAGE electrophoresis byloading 20 μl of the 2 previously boiled samples.

The medium containing the induced bacteria is then centrifuged at7.500×g for 10 min. or at 3000×g for 25 min. at 4° C. and thesupernatant is eliminated.

Results: The induction results are checked by SDS-PAGE electrophoresisas shown in FIG. 1.

EXAMPLE 2 Extraction and Purification of the Inclusion Bodies

The following procedure relates to the preparation and refolding of theinclusion bodies of PlGF-1. By means of refolding the PLGF-1 bacterialprotein is partially brought back to the dimeric form.

Material:

Mixer with appropriate capacity.

Lysis solution: 1 mM Mg2SO4 + 20 mM Tris-HCl pH8 + Triton X100 by 1%.Washing solution: 0.5% triton X100 + 10 mM EDTA pH 8. BD (denaturingbuffer): 8 M urea, 50 mM Tris pH 8, Ethylenediamine 20 mM. Dissolvingand bringing to volume in H2O. Oxidised glutathione 200x: 100 mM in H2O;Reduced glutathione 200x: 250 mM in H2O. Dilution buffer: 600 μM finalPEG 4000 (2.4 g/l), 50 mM Tris-HCl pH 8, 20 mM NaCl. Antifoam: AntifoamO-10 (not siliconic) Sigma.

Preparation of the PLGF-1 Inclusion Bodies.

The lysis and washing solutions are equilibrated at room temperature(RT).

Two cycles of freezing/thawing at −80/37° C. are performed.

The bacterial pellet is lysed in 1 ml of lysis solution per each 450OD600 of bacteria.

It is then incubated at RT 30 min. under stirring (250 RPM).

The solution is poured into a mixer with appropriate capacity and aquantity of washing solution of 3 ml for each 450 OD600 of bacteria isadded.

If necessary, 0.4 μl of not-diluted antifoam per each millilitre ofsample are added.

The solution is spun at the maximum speed for 1 minute or until thesample is well homogeneous.

The content of the mixer is then transferred into a container withappropriate capacity and,6,5 ml of washing solution per each 450 OD600of bacteria are added. It is incubated for 45 min. at RT under stirring.

The so-obtained suspension is centrifuged at 13.000×g for 45 min. at 25°C. and the supernatant is discharged.

The settled pellet is re-suspended in 4 ml of washing solution per each450 OD600 of bacteria and the cycle in the mixer is repeated for thesecond time.

The suspension is transferred into a container with appropriatecapacity, diluted with 6.5 ml of washing solution per each 450 OD600 andincubated for 30 min. at RT under stirring.

The centrifugation under the above seen conditions is then repeated andthe supernatant is eliminated.

EXAMPLE 3 Renaturation of the Protein

The inclusion bodies are solubilised in 7 ml of denaturing buffer BD(containing urea 8M) and further diluted in BD until OD280 of 0.8.Subsequently, 0.6 volumes of dilution buffer are added in order to bringthe final urea concentration to 5M.

Afterwards 1/200 of reduced glutathione 200× (final concentration of1,25 mM) and 1/200 of oxidised glutathione 200× (final concentration of0.5 mM) are added. A 15 μl sample for checking (prerefol) is taken andthe solution is then incubated at 20° C. for 18-20 hours under stirring.

At the end of the incubation, the medium is centrifuged for 10 min. at20° C., 10.000×g, filtered by means of 0.45 or 0.8 μm filters and a 15μl sample is taken for checking (postrefol).

Results: The 15-μl samples of the pre- and post-refolding solutions areanalysed by means of SDS-PAGE electrophoresis (FIG. 5).

EXAMPLE 4 Anion-exchange Chromatography

The following procedure relates to the first step of purification of thePlGF-1 protein after refolding. Upon loading of the sample onto thecolumn, there will be a high loss of not-absorbed PlGF-1 monomer. Theloaded quantity must not exceed 10 times the volumes of the column,since this would cause a significant loss in the PLGF-1 dimer.

The elution is performed under isochratic conditions at 20% of buffer B(see below), which corresponds to a NaCl concentration of 200 mM. Theeluted peak still contains the glutathiones used for refolding, whichcontribute by about 50% of OD280.

Material and parameters:

FPLC system: Amersham-biosciences handled by the software called FPLCDirector. Monitoring parameters U.V.: Wavelength = 280 nm; scale top =2. Temperature: 20° C. (minimum 15, maximum 25) Resin: Q-sepharose FastFlow (Amersham- biosciences) Column volume/height: Volume: 1/10 ofvolume of the sample to be loaded; height: between 13 and 16 cm.Equilibration: 2 Column Volume (CV) of buffer B, then 1.5 CV of bufferA. Sample: Renatured, centrifuged and/or filtrated P1GF-1. Load no morethan 10 CV thereof. Buffer A: 20 mM Ethanolamine-HCl pH 8.5. Buffer B:Buffer A + 1M NaCl. Injection speed: 1 cm/min (maximum speed tested onsmall columns = 1.887 cm/min; minimum tested speed = 0.5 cm/min.).Elution speed: 1 cm/min (maximum speed tested on small columns = 1.887cm/min; minimum tested speed = 0.5 cm/min.). Washings after 1.5 CV with0% of buffer B. Injection: Peak collected: Peak eluted at isochraticconditions at 20% of buffer B, running for about 3 CV. Final washing: 2CV at 100% B.

Procedure:

The peak eluted under isochratic conditions at 20% of buffer B iscollected, then 0.271 water volumes, 0.0045 TFA volumes and 0.225ethanol volumes are added thereto. In this way the sample results to bediluted 1.5 times and contains 15% ethanol and 0.3% TFA. The addition ofthese 2 substances facilitates the bounding of PlGF-1 to thereverse-phase resin (see example 5).

Results: The chromatography step is continuously controlled bymonitoring the optical densities at 280 nm as illustrated by FIG. 2.

The purity of the isolated protein material is analysed by means ofSDS-PAGE electrophoresis (FIG. 5).

EXAMPLE 5 Reverse-phase Chromatography

The following procedure may be performed with RP source resin with 15micron or 30 micron average particle diameter. However, the 30-micron RPsource resin, while not involving any alteration in the purificationprocess, allows an economical saving of the resin itself (about 50%), agreater easiness in the packaging procedure of the column and a lowerbackpressure.

The procedure relates to the second phase of the purification of thePlGF-1 protein after passing on the QFF resin. During the sampleinjection, a high adsorbance is apparent and corresponds to the notadsorbed peak of the glutathiones which do,not bound to the resin. Thisprocedure consists of 2 sub-stages, the, former called RPCmon, is usedto eliminate most of the monomeric component of PlGF-1, whereas thelatter is used to elute the essentially dimeric component of the proteinand it is called RPCdim.

First Sub-stage (RPCmon)

Material and Parameters:

FPLC system: Amersham-biosciences handled by the software called FPLCDirector. Monitoring parameters U.V.: Wavelength = 280 nm; full scale =0.05. Temperature: 20° C. (minimum 15, maximum 25) Resin: Reverse PhaseSource 30 (Amersham- biosciences). Resin volume/height: Volume: ⅕-⅙ ofthe overall OD280 of the sample to be loaded; height: between 27 and 33cm. Equilibration: 2 Column Volume (CV) of buffer B, then 2 CV of bufferA.

Sample: PlGF-1 coming from the preceding step (example 4), diluted 1.5times and containing ethanol 15% and TFA 0.3%. Loading max. 4.5 to 5.5OD280 per ml of resin.

Buffer A: Ethanol 40% + TFA 0.1%. Buffer B: Ethanol 70% + TFA 0.1%.Injection speed: 1.887 cm/min. Elution speed: 1.887 cm/min. Washingsafter 1.5 CV with 4% buffer B. injection:

Monomer peak: Gradient ranging from 4 to 40% of B in 12 CV (3%B/CV).Just after starting the elution of the peak, by OD280 reaching 25% ofthe full scale, the elution is continued with isochratic conditions withthe buffer B concentration reached in that moment until the peak elutionis complete (about 2.5 -3.5 CV).

Operations

A suitable quantity of the solution corresponding to the “monomer” peakis taken and it is concentrated for checking (mon in FIG. 5).

Second Sub-stage (RPCdim)

Material and Parameters:

Without re-equilibrating the column, but by simply shifting the scalerange of the UV monitor to the value 2, a gradient ranging from 10 to100% of the buffer B is run in 2.2 CV (40.9%B/CV).

Operations:

The fractions corresponding to the “Dimer” peak are taken. FIG. 4illustrates an example thereof. They are collected and the volume andthe optical density at 280 nm are measured.

The overall yield (expressed in mg) obtained before the lyophilisationis calculated by multiplying the OD280 times the volume times thedilution. The average value of such yield is 164 mg of pure PLGF-1 perlitre of bacterial culture with a standard deviation of 23.21. It mayalso be expressed in mg per 1000 OD600 of fermented bacteria, resultingto 5.58 mg of pure PLGF-1 each 1000 OD600 of fermented bacteria with astandard deviation of 0.8.

The so-obtained dimer solution is kept at −20° C. untilultradiafiltration and lyophilisation. A sample of such solution issubmitted to SDS-PAGE electrophoresis as illustrated in FIG. 5.

1. A process for extracting and purifying recombinant Placental GrowthFactor (PLGF) protein in dimeric and multimeric active form containingno more than 1.5% of monomeric form, by expression of the PLGF proteinin E. Coli cells modified with an inducible expression system comprisingan insert coding for the PLGF protein, the process comprising in thefollowing order: I) inoculating in a culture medium the E. Coli cellsand fermenting the E. Coli cells until obtaining optical density of theculture medium from 14 to 50 at 600 nm (OD⁶⁰⁰ 14-50); II) inducing theexpression of PLGF protein via the inducible expression system; III)extracting the inclusion bodies by lysing the cultured cells, rupturingDNA of the cultured cells, isolating and purifying the inclusion bodies;IV) solubilizing the inclusion bodies into a denaturing buffer to form asolution and renaturating the expressed protein PLGF by adding to thesolution oxidizing/reducing agents to bring the expressed protein to adimeric form, at least partially; V) passing the solubilized protein ona high flow resin for anion exchange chromatography separating andisolating the dimeric and multimeric forms of the expressed protein fromthe monomeric form; and VI) further separating and finally isolating thedimeric and multimeric forms of the expressed protein from the monomericform by passing them through a reverse-phase chromatography resin withtwo subsequent elution stages with increasing gradient of an organicsolvent separated by an elution stage under isochratic conditions,wherein the organic solvent comprises ethanol, methanol, oracetonitrile, and wherein the monomeric form comprises no more than 1.5%of the total PLGF protein.
 2. The process according to claim 1, whereinthe Placental Growth Factor (PLGF) is human PLGF-1 or of non-humanorigin.
 3. The process according to claim 1, comprising performing thefermentation in a medium comprising at least one selection agents, yeastextract, glycerol and ammonium salts.
 4. The process according to claim1, wherein the inducible expression system is a T7 RNA polymerase IPTGinducible system.
 5. The process according to claim 4, comprisinginducing the expression by adding a compound selected from the groupconsisting of lactose, isopropyl-beta.-D-thiogalactopyranoside (IPTG)and functionally equivalent analogues.
 6. The process according to claim1, wherein the bacterial strain is E. Coli {B121(DE3)pLysS}.
 7. Theprocess according to claim 1, comprising inducing expression when theoptical density of the culture is 14 to 30 OD at 600 nm (14-30 OD⁶⁰⁰).8. The process according to claim 1, comprising performing the celllysis by one of the group consisting of freezing/thawing and Frenchpress.
 9. The process according to claim 1, comprising performing theDNA rupture by one of the group consisting of DNAse of extractionorigin, DNAase of recombinant origin, and chemical-mechanical action.10. The process according to claim 1, comprising performing the DNArupture by mixing with a mixer.
 11. The process according to claim 1,comprising isolating the inclusion bodies by centrifuging for at leasttwo cycles and washing into a suitable buffer.
 12. The process accordingto claim 1, wherein the extracting step further comprises homogenizingor sonicating the solution of inclusion bodies, and wherein thedenaturing buffer comprising a compound selected from the groupconsisting of urea, guanidine isothiocyanate, andguanidine-hydrochloride.
 13. The process according to claim 1, whereinin the renaturing step IV, the solution of the denatured inclusionbodies is incubated with the oxidising/reducing agents for 10 to 30hours at a temperature of 10° C. to 30° C.
 14. The process according toclaim 13, further comprising diluting the solution of denaturedinclusion bodies until obtaining an optical density at 280 nm (OD²⁸⁰)from 0.01 to 2 and adding reduced/oxidised glutathione to form thedimeric form of PLGF protein.
 15. The process according to claim 1,wherein, in step V, said passing is done with a ratio Volumeloaded/Volume column from 1:1 to 10:1.
 16. The process according toclaim 15 wherein the ratio Volume loaded/Volume column is 10:1.
 17. Theprocess according to claim 15, comprising eluting the protein with anethanolamine-HCl, NaCl elution buffer, wherein the protein in monomericform is mainly comprised in the a percolate not bound to the resin ofthe anionic-exchange column, and wherein the protein in essentiallydimeric form is eluted with elution buffers comprising NaCl at aconcentration from 150 to 250 mM.
 18. The process according to claim 17,comprising diluting the eluted fraction in essentially dimeric form andloading the eluted fraction of the protein in essentially dimeric formonto a reverse-phase chromatography column in an amount corresponding toan optical density from 4.5 to 5.5 OD at 280 nm per ml of resin.
 19. Theprocess according to claim 18, additionally comprising performing withfirst and second elution buffers a first elution stage under conditionsof increasing gradient of organic solvent until the elution peak of themonomeric form appears, prosecuting the elution under isochraticconditions until exhaustion of the elution peak of the monomeric form,and performing a second elution stage under conditions of increasinggradient of organic solvent until complete elution of the protein inmainly dimeric form.
 20. The process according to claim 19, wherein thefirst and the second elution buffers comprise a solvent selected fromthe group consisting of ethanol, methanol, and acetonitrile.
 21. Theprocess according to claim 19, wherein the first and the second elutionbuffers are a hydroalcoholic solution comprising increasing percentagesof ethanol.
 22. The process according to claim 18 comprising performingthe reverse-phase chromatography on resin having particles with anaverage diameter of 30-micron.
 23. The process according to claim 1,comprising performing after step IV an additional ultrafiltration stepfollowed by lyophilisation.
 24. A product obtained by the process ofclaim 1, the product comprising not less than 98.5% of Placental GrowthFactor (PLGF) in active dimeric and multimeric form, the monomeric formin a percentage not higher than 1.5%, the product being free frombacterial contaminants.